Combined preparations for the treatment of cancer

ABSTRACT

Combined preparations for the treatment of cancer are described. The combined preparations comprise: (a) LAG-3 protein, or a derivative thereof that is able to bind to MHC class II molecules; and (b) an anti-neoplastic agent, wherein the anti-neoplastic agent is a platinum-based anti-neoplastic agent or a topoisomerase I inhibitor. Methods for the treatment of cancer using the combined preparations are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/105,789, filed Jun. 17, 2016, which is a National Stage Entry ofInternational Application No. PCT/EP2014/078779, filed Dec. 19, 2014,which claims priority from GB 1322626.1, filed on Dec. 19, 2013, theentire contents of which are incorporated herein by reference herein.

This invention relates to combined preparations and to pharmaceuticalcompositions, and their use as medicaments, in particular for thetreatment of cancer, and to methods for the treatment of cancer.

Cancer may be treated with one or more cytotoxic anti-neoplastic drugs(“chemotherapeutic agents”) as part of a standardized regimen.Chemotherapy may be aimed at curing a patient, or at prolonging life, oralleviating symptoms.

Conventional chemotherapeutic agents act by killing cells that dividerapidly, exploiting one of the properties of most cancer cells. However,chemotherapy also harms cells that divide rapidly under normalcircumstances, for example cells in the bone marrow, digestive tract,and hair follicles. This causes the most common side-effects ofchemotherapy: myelosuppression (decreased production of blood cells,hence also immunosuppression), mucositis (inflammation of the lining ofthe digestive tract), and alopecia (hair loss).

There is a need to provide more effective cancer treatments, and toprovide effective cancer treatments with reduced side effects.

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog type Imembrane protein with four extracellular Ig superfamily domains. Similarto CD4, LAG-3 oligomerizes at the surfaces of T cells and binds to MHCclass II molecules on antigen-presenting cells (APCs) but withsignificantly higher affinity than CD4. LAG-3 is expressed on activatedCD4-positive and CD8-positive T lymphocytes where it associates with theCD3-TCR complex at the cell surface and negatively regulates signaltransduction. As a consequence, it negatively regulates T cellproliferation, function, and homeostasis.

LAG-3-derived soluble fusion proteins, have been shown to bind MHC classII molecules with a much higher avidity than CD4, to increase thecapacity of MHC class II-positive macrophages and immature dendriticcells to induce T cell responses in vitro, and to enhance the in vitroinduction of viral and tumor-specific cytotoxic T cells. Accordingly, aLAG-3 fusion protein is used as a systemic immunostimulant and as anadjuvant for cancer vaccines.

WO 2009/044273 describes use of recombinant LAG-3 protein, orderivatives thereof, for boosting a monocyte-mediated immune response,in particular to induce an increase in the number of monocytes in blood,for the treatment of cancer.

It has now surprisingly been found that administration of LAG-3 protein,or a derivative thereof that is able to bind to MHC class II molecules,and a platinum-based anti-neoplastic agent, or a topoisomerase Iinhibitor, has a synergistic effect on reducing tumor growth.

According to the invention there is provided a combined preparation,which comprises: (a) LAG-3 protein, or a derivative thereof that is ableto bind to MHC class II molecules; and (b) an anti-neoplastic agent,wherein the anti-neoplastic agent is a platinum-based anti-neoplasticagent or a topoisomerase I inhibitor.

The term “combined preparation” as used herein refers to a “kit ofparts” in the sense that the combination components (a) and (b) asdefined above can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination components(a) and (b). The components can be administered simultaneously or oneafter the other. If the components are administered one after the other,preferably the time interval between administration is chosen such thatthe therapeutic effect of the combined use of the components is greaterthan the effect which would be obtained by use of only any one of thecombination components (a) and (b).

The components of the combined preparation may be present in onecombined unit dosage form, or as a first unit dosage form of component(a) and a separate, second unit dosage form of component (b). The ratioof the total amounts of the combination component (a) to the combinationcomponent (b) to be administered in the combined preparation can bevaried, for example in order to cope with the needs of a patientsub-population to be treated, or the needs of the single patient, whichcan be due, for example, to the particular disease, age, sex, or bodyweight of the patient.

Preferably, there is at least one beneficial effect, for example anenhancing of the effect of the anti-neoplastic agent, or a mutualenhancing of the effect of the combination components (a) and (b), forexample a more than additive effect, additional advantageous effects,fewer side effects, less toxicity, or a combined therapeutic effectcompared with an effective dosage of one or both of the combinationcomponents (a) and (b), and very preferably a synergism of thecombination components (a) and (b).

A combined preparation of the invention may be provided as apharmaceutical combined preparation for administration to a mammal,preferably a human. The LAG-3 protein, or derivative thereof, mayoptionally be provided together with a pharmaceutically acceptablecarrier, excipient, or diluent, and/or the anti-neoplastic agent mayoptionally be provided together with a pharmaceutically acceptablecarrier, excipient, or diluent.

The LAG-3, or derivative thereof, may be present at a dose which is amolar equivalent of 0.25-30 mg, 1-30 mg, or 6-30 mg of the LAG-3derivative LAG-3Ig fusion protein IMP321. Doses of 6-30 mg persubcutaneous (s.c.) injection of IMP321 have been shown to be safe andprovide an acceptable systemic exposure based on the results ofpharmacokinetics data obtained in metastatic renal cell cancer patients.A blood concentration of IMP321 superior to 1 ng/ml for at least 24hours after s.c. injection is obtained in patients injected with IMP321doses of more than 6 mg.

A combined preparation of the invention may comprise a plurality ofdoses of the LAG-3 protein, or derivative thereof.

The dose of the anti-neoplastic agent will depend on the particularanti-neoplastic agent being used.

Platinum-based anti-neoplastic agents are coordination complexes ofplatinum used in cancer chemotherapy. They are believed to formcross-links in DNA that inhibit DNA repair and/or DNA synthesisresulting in cell death. The main dose-limiting side effect of cancertreatment using platinum compounds is peripheral neurotoxicity. Examplesof platinum-based anti-neoplastic agents include cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, Nedaplatin, and Triplatin.

Carboplatin, orcis-diammine(cyclobutane-1,1-dicarboxylate-O,O′)platinum(I) (trade namesParaplatin and Paraplatin-AQ), is used against some forms of cancer(mainly ovarian carcinoma, lung, head and neck cancers as well asendometrial, esophageal, bladder, breast and cervical; central nervoussystem or germ cell tumors; osteogenic sarcoma, and as preparation for astem cell or bone marrow transplant). It has greatly reducedside-effects compared to its parent compound cisplatin. Guidelines forcarboplatin dosing are available from the US Food and DrugAdministration (FDA).

Oxaliplatin, or[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II) (tradename Eloxatin), comprises a square planar platinum(II) centre. Incontrast to cisplatin and carboplatin, oxaliplatin comprises thebidentate ligand 1,2-diaminocyclohexane in place of the two monodentateamine ligands. It also has a bidentate oxalate group.

Oxaliplatin has anti-tumor activity against colon carcinoma. Oxaliplatinfunctions by forming both inter- and intra-strand cross links in DNA.Cross links in DNA prevent DNA replication and transcription, resultingin cell death. The recommended dose of oxaliplatin in an adjuvantsetting is 85 mg/m² intravenously repeated every two weeks for 12cycles. A recommended dose for oxaliplatin in treatment of metastaticcolorectal cancer is 85 mg/m² intravenously repeated every two weeksuntil disease progression or unacceptable toxicity.

Topoisomerase inhibitors are agents designed to interfere with theaction of topoisomerase enzymes (topoisomerase I and II), which areenzymes that control changes in DNA structure by catalyzing the breakingand rejoining of the phosphodiester backbone of DNA strands during thenormal cell cycle. It is thought that topoisomerase inhibitors block theligation step of the cell cycle, generating single and double strandedbreaks that harm the integrity of the genome. Introduction of thesebreaks subsequently leads to apoptosis and cell death.

Human DNA topoisomerase I (Top1) is an essential enzyme that relaxes DNAsupercoiling during replication and transcription. Top1 generates DNAsingle-strand breaks that allow rotation of the cleaved strand aroundthe double helix axis. Top1 also re-ligates the cleaved strand tore-establish intact duplex DNA. Top1-DNA intermediates, known ascleavage complexes, are transient and present at low levels under normalcircumstances. However, treatment with Top1 inhibitors, such as thecamptothecins, stabilizes the cleavable complexes, prevents DNAreligation and induces lethal DNA strand breaks. Cancer cells areselectively sensitive to the generation of these DNA lesions.

Topotecan, or(S)-10-[(dimethylamino)methy]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione monohydrochloride (trade name Hycamtin), isa chemotherapeutic agent that is a topoisomerase I inhibitor. It is awater-soluble derivative of camptothecin. It is used in form of thehydrochloride to treat ovarian cancer and lung cancer, as well as othercancer types. Topotecan is a semi-synthetic derivative of camptothecin.Camptothecin is a natural product extracted from the bark of the treeCamptotheca acuminata. Topoisomerase-I is a nuclear enzyme that relievestorsional strain in DNA by opening single strand breaks. Oncetopoisomerase-I creates a single strand break, the DNA can rotate infront of the advancing replication fork. Topotecan intercalates betweenDNA bases. This intercalation disrupts the DNA duplication machinerywhen it reaches a site where topotecan is intercalated. This disruptionprevents DNA replication, and ultimately leads to cell death. Mammaliancells cannot efficiently repair these double strand breaks. This processleads to breaks in the DNA strand resulting in apoptosis.

A recommended dose of Hycamtin capsules is 2.3 mg/m² body surfacearea/day administered for five consecutive days with a three weekinterval between the start of each course.

Another camptothecin derivative irinotecan (CPT11) is approved for thetreatment of colon cancer.

A combined preparation of the invention may comprise a plurality ofdoses of the anti-neoplastic agent.

The LAG-3 protein may be an isolated natural or recombinant LAG-3protein. The LAG-3 protein may comprise an amino sequence of LAG-3protein from any suitable species, such as a primate or murine LAG-3protein, but preferably a human LAG-3 protein. The amino acid sequenceof human and murine LAG-3 protein is provided in FIG. 1 of Huard et al(Proc. Natl. Acad. Sci. USA, 11: 5744-5749, 1997). The sequence of humanLAG-3 protein is repeated in FIG. 13 below (SEQ ID NO: 1). The aminoacid sequences of the four extracellular Ig superfamily domains (D1, D2,D3, and D4) of human LAG-3 are also identified in FIG. 1 of Huard etal., at amino acid residues: 1-149 (D1); 150-239 (D2); 240-330 (D3); and331-412 (D4).

Derivatives of LAG-3 protein include fragments, variants, or mutants ofLAG-3protein that are able to bind MHC class II molecules. Severalderivatives of LAG-3 protein are known that are able to bind to MHCclass II molecules. Many examples of such derivatives are described inHuard et al (Proc. Natl. Acad. Sci. USA, 11: 5744-5749, 1997). Thisdocument describes characterization of the MHC class II binding site onLAG-3 protein. Methods for making mutants of LAG-3 are described, aswell as a quantitative cellular adhesion assay for determining theability of LAG-3 mutants to bind class II-positive Daudi cells. Bindingof several different mutants of LAG-3 to MHC class II molecules wasdetermined. Some mutations were able to reduce class II binding, whileother mutations increased the affinity of LAG-3 for class II molecules.Many of the residues essential for binding MHC class II proteins areclustered at the base of a large 30 amino acid extra-loop structure inthe LAG-3 D1 domain. The amino acid sequence of the extra-loop structureof the D1 domain of human LAG-3 protein isGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 2), the underlined sequencein FIG. 13.

The LAG-3 protein derivative may comprise the 30 amino acid extra-loopsequence of the human LAG-3 D1 domain, or a variant of such sequencewith one or more conservative amino acid substitutions. The variant maycomprise amino acid sequence that has at least 70%, 80%, 90%, or 95%amino acid identity with the 30 amino acid extra-loop sequence of thehuman LAG-3 D1 domain.

The derivative of LAG-3 protein may comprise an amino acid sequence ofdomain D1, and optionally domain D2, of LAG-3 protein, preferably humanLAG-3 protein.

The derivative of LAG-3 protein may comprise an amino acid sequence thathas at least 70%, 80%, 90%, or 95% amino acid identity with domain D1,or with domain D1 and D2, of LAG-3 protein, preferably human LAG-3protein.

The derivative of LAG-3 protein may comprise an amino acid sequence ofdomains D1, D2, D3, and optionally D4, of LAG-3 protein, preferablyhuman LAG-3 protein.

The derivative of LAG-3 protein may comprise an amino acid sequence thathas at least 70%, 80%, 90%, or 95% amino acid identity with domain D1,D2, and D3, or with domain D1, D2, D3, and D4, of LAG-3 protein,preferably human LAG-3.

Sequence identity between amino acid sequences can be determined bycomparing an alignment of the sequences. When an equivalent position inthe compared sequences is occupied by the same amino acid, then themolecules are identical at that position.

Scoring an alignment as a percentage of identity is a function of thenumber of identical amino acids at positions shared by the comparedsequences. When comparing sequences, optimal alignments may require gapsto be introduced into one or more of the sequences to take intoconsideration possible insertions and deletions in the sequences.Sequence comparison methods may employ gap penalties so that, for thesame number of identical molecules in sequences being compared, asequence alignment with as few gaps as possible, reflecting higherrelatedness between the two compared sequences, will achieve a higherscore than one with many gaps. Calculation of maximum percent identityinvolves the production of an optimal alignment, taking intoconsideration gap penalties.

Suitable computer programs for carrying out sequence comparisons arewidely available in the commercial and public sector. Examples includeMatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; programavailable from http://bitincka.com/ledion/matgat), Gap (Needleman &Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990,J. Mol. Biol. 215: 403-410; program available fromhttp://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al.,2007, Bioinformatics 23: 2947-2948; program available fromhttp://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise AlignmentAlgorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Timewarps, string edits and macromolecules: the theory and practice ofsequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley:programs available from http://www.ebi.ac.uk/tools/emboss/align). Allprograms may be run using default parameters.

For example, sequence comparisons may be undertaken using the “needle”method of the EMBOSS Pairwise Alignment Algorithms, which determines anoptimum alignment (including gaps) of two sequences when considered overtheir entire length and provides a percentage identity score. Defaultparameters for amino acid sequence comparisons (“Protein Molecule”option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix:Blosum 62.

The sequence comparison may be performed over the full length of thereference sequence.

The LAG-3 protein derivative may be fused to Immunoglobulin Fc aminoacid sequence, preferably human IgG1 Fc amino acid sequence, optionallyby a linker amino acid sequence.

The ability of a derivative of LAG-3 protein to bind to MHC class IImolecules may be determined using a quantitative cellular adhesion assayas described in Huard et al (supra). The affinity of a derivative ofLAG-3 protein for MHC class II molecules may be at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% of the affinity of human LAG-3 proteinfor class II molecules. Preferably the affinity of a derivative of LAG-3protein for MHC class II molecules is at least 50% of the affinity ofhuman LAG-3 protein for class II molecules.

-   -   Examples of suitable derivatives of LAG-3 protein that are able        to bind MHC class II molecules include derivatives comprising:    -   amino acid residues 23 to 448 of the human LAG-3 sequence;    -   amino acid sequence of domains D1 and D2 of LAG-3;    -   amino acid sequence of domains D1 and D2 of LAG-3 with an amino        acid substitution at one or more of the following positions:        position 73 where ARG is substituted with GLU; position 75 where        ARG is substituted with ALA or GLU; position 76 where ARG is        substituted with GLU; position 30 where ASP is substituted with        ALA; position 56 where HIS is substituted with ALA: position 77        where TYR is substituted with PHE; position 88 where ARG is        substituted with ALA; position 103 where ARG is substituted with        ALA; position 109 where ASP is substituted with GLU; position        115 where ARG is substituted with ALA;    -   amino acid sequence of domain D1 of LAG-3 with a deletion of        amino acid residues 54 to 66;    -   a recombinant soluble human LAG-3Ig fusion protein (IMP321)—a        200-kDa dimer produced in Chinese hamster ovary cells        transfected with a plasmid encoding for the extracellular domain        of hLAG-3 fused to the human IgG1 Fc.

According to the invention there is also provided a pharmaceuticalcomposition, which comprises (a) LAG-3 protein, or a derivative thereofthat is able to bind to MHC class II molecules; (b) an anti-neoplasticagent, wherein the anti-neoplastic agent is a platinum-basedanti-neoplastic agent or a topoisomerase I inhibitor; and (c) apharmaceutically acceptable carrier, excipient, or diluent.

According to the invention there is further provided a combinedpreparation, or pharmaceutical composition, of the invention for use asa medicament.

The invention also provides a combined preparation, or pharmaceuticalcomposition, of the invention for preventing, treating, or amelioratingcancer.

There is further provided according to the invention use of a combinedpreparation, or pharmaceutical composition, of the invention in themanufacture of a medicament for preventing, treating, or amelioratingcancer.

There is also provided according to the invention a method ofpreventing, treating, or ameliorating cancer, which comprisesadministering LAG-3 protein, or a derivative thereof that is able tobind to MHC class II molecules, and an anti-neoplastic agent, to asubject in need of such prevention, treatment, or amelioration, whereinthe anti-neoplastic agent is a platinum-based anti-neoplastic agent or atopoisomerase 1 inhibitor.

The LAG-3 protein, or derivative thereof, and the anti-neoplastic agentmay be administered sequentially to the subject, i.e. the LAG-3 protein,or derivative thereof, may be administered before, with, or after theanti-neoplastic agent.

The LAG-3 protein, or derivative thereof, and the anti-neoplastic agentmay be administered to the subject within 96 hours, 72 hours, 48 hours,24 hours, or 12 hours, of each other.

Alternatively, the LAG-3 protein, or derivative thereof, and theanti-neoplastic agent may be co-administered to the subject, for exampleas a composition comprising the LAG-3 protein, or derivative thereof,and the anti-neoplastic agent, or by simultaneous administration ofseparate doses of the LAG-3 protein, or derivative thereof, and theanti-neoplastic agent.

According to some embodiments, a plurality of doses of the LAG-3protein, or derivative thereof, and/or a plurality of doses of theanti-neoplastic agent, is administered to the subject.

According to some embodiments, a dose of the LAG-3 protein, orderivative thereof, is administered before, with, or after eachadministration of two or more doses of the anti-neoplastic agent.

For example, a dose of the LAG-3 protein, or derivative thereof, may beadministered within 96 hours, 72 hours, 48 hours, 24 hours, or 12 hours,of each administration of two or more doses of the anti-neoplasticagent.

The choice of appropriate dosages of the components used in combinationtherapy according to the present invention can be determined andoptimized by the skilled person, for example, by observation of thepatient, including the patient's overall health, and the response to thecombination therapy. Optimization, for example, may be necessary if itis determined that a patient is not exhibiting the desired therapeuticeffect or conversely, if the patient is experiencing undesirable oradverse side effects that are too many in number or are of a troublesomeseverity.

The doses of the components used in combination therapy according to theinvention should be chosen to provide a therapeutically effective amountof the components in combination. An “effective amount” of thecombination therapy is an amount that results in a reduction of at leastone pathological parameter associated with cancer. For example, in someembodiments, an effective amount of the combination therapy is an amountthat is effective to achieve a reduction of at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90%, in the parameter, compared to theexpected reduction in the parameter associated with the cancer withoutthe combination therapy. For example, the parameter may be tumor growth.

According to the invention, combination treatment may be employed toincrease the therapeutic effect of the anti-neoplastic agent, or LAG-3protein, or derivative thereof, compared with the effect of theanti-neoplastic agent, or LAG-3 protein, or derivative thereof, as amonotherapy, or to decrease the doses of the individual components inthe resulting combinations while preventing or further reducing the riskof unwanted or harmful side effects of the individual components.

In one embodiment, the LAG-3 protein, or derivative thereof, and theanti-neoplastic agent are each prescribed at a dose that is within atypically prescribed dose range for each compound as a monotherapy. Thecompounds may be prescribed as separate dosages or as a combinationdosage. Such combinations provide increased efficacy compared with theeffect of either compound as a monotherapy.

In another embodiment, the LAG-3 protein, or derivative thereof, and theanti-neoplastic agent are each prescribed at a dose that is below atypically prescribed dose for each component as a monotherapy, but atdoses that have therapeutic efficacy in combination. The components maybe prescribed as separate dosages or as a combination dosage. Thedosages of the components in combination may be selected to provide asimilar level of therapeutic efficacy as the LAG-3 protein, orderivative thereof, or the anti-neoplastic agent as a monotherapy, butwith the advantage that the lower doses of the LAG-3 protein, orderivative thereof, and the anti-neoplastic agent reduce the risk ofadverse side effects compared to the prescribed dosages of each compoundas a monotherapy.

In another embodiment, the prescribed dosage of the anti-neoplasticagent is within a typically prescribed dose range for monotherapy, andthe LAG-3 protein, or derivative thereof, is prescribed at a dosage thatis below a typically prescribed dose for monotherapy.

In a further embodiment, the prescribed dosage of the anti-neoplasticagent is below a typically prescribed dose for monotherapy, and theLAG-3 protein, or derivative thereof, is prescribed at a dosage that iswithin a typically prescribed dose range for monotherapy.

Preferred dosages below the typically prescribed dose for monotherapyare doses that are up to 50%, or up to 25%, of the typically prescribeddose.

When administered in separate dosages, the LAG-3 protein, or derivativethereof, and the anti-neoplastic agent may be administered substantiallysimultaneously (for example, within about 60 minutes, about 50 minutes,about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes,about 5 minutes, or about 1 minute of each other) or separated in timeby about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10hours, about 12 hours, about 24 hours, about 36 hours, about 72 hours,or about 96 hours, or more.

The skilled person will be able to determine, and optimise, a suitabletime course for sequential administration, depending on the particularcombination of the LAG-3 protein, or derivative thereof, and theanti-neoplastic agent. The time course is preferably selected such thatthere is at least one beneficial effect, for example an enhancing of theeffect of the LAG-3 protein, or derivative thereof, or theanti-neoplastic agent, or a mutual enhancing of the effect of thecombination components, for example a more than additive effect,additional advantageous effects, fewer side effects, less toxicity, or acombined therapeutic effect compared with a non-effective dosage of oneor both of the combination components, and very preferably a synergismof the combination components.

It will be appreciated that the optimum time course will depend on thefactors such as the time taken for the peak plasma concentration of thecompound to be reached after administration, and the eliminationhalf-life of each compound. Preferably the time difference is less thanthe half-life of the first component to be administered.

The skilled person will also be able to determine appropriate timing foradministration. In certain embodiments, the anti-neoplastic agent may beadministered in the morning, and the LAG-3 protein, or derivativethereof, administered at least once later in the day. In otherembodiments, the anti-neoplastic agent and LAG-3 protein, or derivativethereof, may be administered at substantially the same time.

In some embodiments, the anti-neoplastic agent may be administered tothe subject, for example, by a medical practitioner, and the subject maybe provided with a dose of the LAG-3 protein, or derivative thereof, forexample in a pre-filled syringe, to administer later (for example laterthe same day, or the next day).

The subject may receive doses of the anti-neoplastic agent and LAG-3protein, or derivative thereof, over a period of weeks, months, oryears. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more.

Preferably the subject is a mammalian subject, more preferably a humansubject.

Examples of cancers that may be treated according to the inventioninclude breast, ovarian, lung, head, neck, endometrial, esophageal,bladder, cervical, osteogenic sarcoma, colon, colorectal cancer,lymphoma, and central nervous system or germ cell tumors.

In general, the components of a combination of the invention, or acomposition of the invention, may be administered by known means, in anysuitable formulation, by any suitable route. In some embodiments, theLAG-3 protein, or derivative thereof, is administered parenterally(including by subcutaneous, intravenous, or intramuscular injection). Insome embodiments, the anti-neoplastic agent is administeredintravenously. In particular embodiments, the LAG-3 protein, orderivative thereof, is administered subcutaneously, and theanti-neoplastic agent is administered intravenously.

Suitable pharmaceutical compositions and dosage forms may be preparedusing conventional methods known to those in the field of pharmaceuticalformulation and described in the relevant texts and literature, forexample, in Remington: The Science and Practice of Pharmacy (Easton,Pa.: Mack Publishing Co., 1995).

It is especially advantageous to formulate combinations or compositionsof the invention in unit dosage form for ease of administration anduniformity of dosage. The term “unit dosage forms” as used herein refersto physically discrete units suited as unitary dosages for theindividuals to be treated. That is, the compositions are formulated intodiscrete dosage units each containing a predetermined, “unit dosage”quantity of an active agent calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specifications of unit dosage forms of the invention aredependent on the unique characteristics of the active agent to bedelivered. Dosages can further be determined by reference to the usualdose and manner of administration of the ingredients. It should be notedthat, in some cases, two or more individual dosage units in combinationprovide a therapeutically effective amount of the active agent, forexample, two tablets or capsules taken together may provide atherapeutically effective dosage, such that the unit dosage in eachtablet or capsule is approximately 50% of the therapeutically effectiveamount.

Preparations according to the invention for parenteral administrationinclude sterile aqueous and non-aqueous solutions, suspensions, andemulsions. Injectable aqueous solutions contain the active agent inwater-soluble form. Examples of non-aqueous solvents or vehicles includefatty oils, such as olive oil and corn oil, synthetic fatty acid esters,such as ethyl oleate or triglycerides, low molecular weight alcoholssuch as propylene glycol, synthetic hydrophilic polymers such aspolyethylene glycol, liposomes, and the like. Parenteral formulationsmay also contain adjuvants such as solubilizers, preservatives, wettingagents, emulsifiers, dispersants, and stabilizers, and aqueoussuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, anddextran. Injectable formulations may be rendered sterile byincorporation of a sterilizing agent, filtration through abacteria-retaining filter, irradiation, or heat. They can also bemanufactured using a sterile injectable medium. The active agent mayalso be in dried, e.g., lyophilized, form that may be rehydrated with asuitable vehicle immediately prior to administration via injection.

In addition to the formulations described previously, the active agentmay be formulated as a depot preparation for controlled release of theactive agent, preferably sustained release over an extended time period.These sustained release dosage forms are generally administered byimplantation (for example, subcutaneously or intramuscularly or byintramuscular injection).

Combined preparations of the invention may be packaged with instructionsfor administration of the components on the combination. Theinstructions may be recorded on a suitable recording medium orsubstrate. For example, the instructions may be printed on a substrate,such as paper or plastic. The instructions may be present as a packageinsert, in the labeling of the container or components thereof (i.e.,associated with the packaging or sub-packaging). In other embodiments,the instructions are present as an electronic storage data file presenton a suitable computer readable storage medium, for example, CD-ROM,diskette. Some or all components of the combined preparation may bepackaged in suitable packaging to maintain sterility.

Embodiments of the invention are described in the examples below, withreference to the accompanying drawings in which:

FIG. 1 shows the effect of administration of a LAG-3 derivative andTopotecan in the treatment of cancer;

FIG. 2 shows the effect of administration of a LAG-3 derivative andCarboplatin in the treatment of cancer;

FIGS. 3A-3B shows the effect of administration of (A) a LAG-3 derivativeand Carboplatin, or (B) a LAG-3 derivative and Oxaliplatin in thetreatment of cancer;

FIGS. 4A-4B shows the effect of administration of a LAG-3 derivative andOxaliplatin in the treatment of cancer: (A) shows the effect on tumorsize, and (B) shows the effect on survival;

FIG. 5 shows an illustration of derivatives of LAG-3 protein fused toImmunoglobulin Fc (IgFc) sequence;

FIG. 6 shows binding of LAG-3 derivatives to MHC class II-positivecells:

FIG. 7 shows inhibition of binding of a LAG-3 derivative to MHC classII-positive cells by antibodies that block binding of LAG-3 to MHC classII molecules;

FIG. 8 shows activation of THP-1 cells by LAG-3 derivatives, asdetermined by CCL4 secretion;

FIG. 9 shows activation of THP-1 cells by LAG-3 derivatives, asdetermined by TNF-α secretion;

FIG. 10 shows inhibition of LAG derivative-induced monocyte activationby antibodies that block binding of LAG-3 to MHC class II molecules;

FIG. 11 shows activation of antigen-presenting cells (APCs) by LAG-3derivatives;

FIG. 12 shows activation of CD8-positive T cells by LAG-3 derivatives;and

FIG. 13 shows amino acid sequence of mature human LAG-3 protein. Thefour extracellular Ig superfamily domains are at amino acid residues:1-149 (D1); 150-239 (D2); 240-330 (3); and 331-412 (04). The amino acidsequence of the extra-loop structure of the D1 domain of human LAG-3protein is shown underlined in bold.

EXAMPLE 1

Effect of Administration of a LAG-3 Derivative and a Topolsomerase IInhibitor in the Treatment of Cancer

A murine syngeneic skin tumour model was established using thecolorectal adenocarcinoma cell line CT26.

One quarter of the minimum tumorigenic dose (MTD) of tumour cells(0.5×10⁵ cells) was implanted by subcutaneous (s.c.) injection in theright flank of four groups of BALB/c mice (5 weeks old) at Day 0. Themice were injected with phosphate buffered saline (PBS) (Group 1 mice),the LAG-3 derivative IMP321 (Group 2 mice), IMP321 and Topotecan (Group3 mice), or Topotecan alone (Group 4 mice) according to the followingschedule:

-   -   Group 1 (8 mice): negative control: PBS s.c. injection at D11,        D14, D18, D21, D25 and D28;    -   Group 2 (7 mice): IMP321 s.c. injection (50 μg, 1.9 mg/ml) at        D11, D14, D18, D21, D25 and D28;    -   Group 3 (8 mice): IMP321 s.c. injection (50 μg, 1.9 mg/ml) at        D11, D14, D18, D21, D25 and D28, plus Topotecan i.p. injection        (45 μg, 2.5 mg/kg) at D10, D13 and D17;    -   Group 4 (8 mice): Topotecan i.p. injection (45 μg, 2.5 mg/kg) at        D10, D13 and D17.

Tumour growth was monitored twice a week by measuring two perpendiculartumour diameters using calipers. The results are shown in FIG. 1. Tumoursize means the cross-sectional area in mm².

The results show that IMP321 alone had no effect on tumour growth,topotecan had some effect on reducing tumor growth, but combinedtreatment with IMP321 and topotecan has a greater (i.e. a synergistic)effect.

EXAMPLE 2

Effect of Administration of a LAG-3 Derivative and a Platinum-BasedAnti-Neoplastic Agent in the Treatment of Cancer

A murine syngeneic skin tumour model was established using the lymphomacell line EL4.

The minimum tumorigenic dose (MTD) of tumour cells (5×10⁵ cells) wasimplanted by s.c. injection in the right flank of C57Bl/6 mice (5 weeksold) at Day 0. The mice were injected with phosphate buffered saline(PBS) (Group 1 mice), IMP321 (Group 2 mice), IMP321 and Carboplatin(Group 3 mice), or Carboplatin alone (Group 4 mice) according to thefollowing schedule:

-   -   Group 1 (8 mice): negative control: PBS s.c. injection at D7,        D11, D14, D19, D21 and D24;    -   Group 2 (8 mice): IMP321 s.c. injection (50 μg, 3.96 mg/ml) at        D7, D11, D14, D19, D21 and D24;    -   Group 3 (8 mice): IMP321 s.c. injection (50 μg, 3.96 mg/m) at        D7, D11, D14, D19, D21 and D24, plus Carboplatin i.p. injection        (288 μg, 16 mg/kg) at D6, D10, D13 and D17;    -   Group 4 (8 mice): Carboplatin i.p. injection (288 μg, 16 mg/kg)        at D6, D10, D13 and D17.

Tumour growth was monitored twice a week by measuring two perpendiculartumor diameters using calipers. The results are shown in FIG. 2. Tumoursize means the cross-sectional area in mm².

The results show that IMP321 alone had no effect on tumour growth,carboplatin alone had very little effect, if any, but combined treatmentwith IMP321 and carboplatin reduced tumour growth, thereby demonstratinga synergistic effect of the combined administration.

EXAMPLE 3

Effect of Administration of a LAG-3 Derivative and DifferentPlatinum-Based Anti-Neoplastic Agents in the Treatment of Cancer

A murine syngeneic skin tumour model was established using the lymphomacell line EL4.

The minimum tumorigenic dose (MTD) of tumour cells (5×10⁵ cells) wasimplanted by s.c. injection in the right flank of C57Bl/6 mice (5 weeksold) at Day 0. The mice were injected with phosphate buffered saline(PBS) (Group 1 mice), IMP321 (Group 2 mice), IMP321 and Carboplatin(Group 3 mice), Carboplatin alone (Group 4 mice), IMP321 and Oxaliplatin(Group 5 mice), or Oxaliplatin alone (Group 6 mice) according to thefollowing schedule:

-   -   Group 1 (10 mice): negative control: PBS s.c. injection at D7,        D11, D14, D18, D21 and D25;    -   Group 2 (10 mice): IMP321 s.c. injection (50 μg, 1.9 mg/m) at        D7, D11, D14, D18, D21 and D25;    -   Group 3 (9 mice): IMP321 s.c. injection (50 μg, 1.9 mg/ml) at        D7, D11, D14, D18, D21 and D25, plus Carboplatin i.p. injection        (288 μg, 16 mg/kg) at D6, D10, D13 and D17;    -   Group 4 (10 mice): Carboplatin i.p. injection (288 μg, 16 mg/kg)        at D6, D10, D13 and D17;    -   Group 5 (10 mice): IMP321 s.c. injection (50 μg, 1.9 mg/m) at        D7, D11, D14, D18, D21 and D25, plus Oxaliplatin i.p. injection        (54 μg, 3 mg/kg) at D6 and D10;    -   Group 6 (10 mice): Oxaliplatin i.p. injection (54 μg, 3 mg/kg)        at D6 and D10.

Tumour growth was monitored twice a week by measuring two perpendiculartumor diameters using calipers. The results are shown in FIG. 3A (forCarboplatin) and FIG. 3B (for Oxaliplatin). Tumour size means thecross-sectional area in mm².

The results show that IMP321 alone had little, if any effect,carboplatin alone had some effect, and combined treatment with IMP321and carboplatin had a greater (i.e. a synergistic) effect. Oxaliplatinalone had an effect, but combined treatment with IMP321 and oxaliplatinhad an even greater (i.e. a synergistic) effect, with tumour growthcompletely inhibited by Day 17.

EXAMPLE 4

Effect of Administration of a LAG-3 Derivative and a Platinum-BasedAnti-Neoplastic Agent in the Treatment of Cancer

The effect of combined treatment with the LAG-3 derivative IMP321 (alsoreferred to as hLAG-3Ig) and oxaliplatin in the C38 colon adenocarcinomatumor model was evaluated. In this model, tumor fragments are surgicallyimplanted subcutaneously. When treatment is begun (at day 12, when themean tumor volume is 200 mm), the tumor is relatively mature and soprovides a good model for real-life tumors.

Mouse colon 38 (C38) tumor fragments were obtained frozen from theDivision of Cancer Treatment, Tumor Repository, NCI (Frederick, Md.,USA). The C38 fragments were stored frozen in DMSO/SVF/RPMI 1640 medium(10/10/80) in liquid nitrogen until use. The fragments were thawed at37° C. for 5 min, rinsed twice in RPMI 1640 medium before subcutaneous(SC) implantation in mice. The C38 tumors were serially transplanted inC57Bl/6 mice.

Small C38 tumor fragments (20-30 mg) were subcutaneously implanted inthe right flank of 12 C57BL/6 mice. When tumor sizes reached 500-1000mm³, tumors were surgically excised and small C38 tumor fragments (20-30mg) were subcutaneously implanted in the right flank of recipientC57BL/6 mice.

Treatment started when the tumors reached a mean volume of 200-300 mm³.The treatment schedule was as follows:

-   -   Group 1 (10 mice): one weekly SC injection of PBS for 4        consecutive weeks;    -   Group 2 (10 mice): one weekly IV injection of oxaliplatin at 5        mg/kg/inj for 4 consecutive weeks;    -   Group 3 (10 mice): one weekly SC injection of 20 μg IMP321 for 4        consecutive weeks:    -   Group 4 (10 mice): one weekly IV injection of oxaliplatin at 5        mg/kg/inj in combination with one weekly SC injection of 20 μg        IMP321 for 4 consecutive weeks.

Treatment started at day 12 (D12) when the different groups had a meantumor volume of 200 mm³. PBS or Oxaliplatin was injected at D12, D19,D26 and D33. IMP321 was injected the day after Oxaliplatin, that is atD13, D20, 27 and D34. Animals were terminated when the subcutaneoustumor reached a maximum volume of 2,000 mm³:

Administration Treatment Group Treatment Dose route schedule 1 PBS — SCQ7Dx4 2 Oxaliplatin 5 mg/kg/inj IV Q7Dx4 3 IMP321 20 μg/mouse/inj SCQ7Dx4* 4 Oxaliplatin 5 mg/kg/inj IV Q7Dx4 IMP321 20 μg/mouse/inj SCQ7Dx4* *performed the day after the treatment with oxaliplatin

The results are shown in FIG. 4A. The results show that IMP321 alone hadno effect on delaying tumor growth. Oxaliplatin had a slight effect. Thecombination of Oxaliplatin and IMP321 had a greater effect. The samesynergistic effect is seen in the survival curves, shown in FIG. 4B.

EXAMPLE 5

Binding of LAG-3 Derivatives to MHC Class II-Positive Cells

Several derivatives of LAG-3 were tested for their ability to bind toMHC class II-positive cells:

i) domains D1-D4 of LAG-3 linked to immunoglobulin Fc (Ig Fc) sequenceby a first linker (LAG-3 D1D4-linker1-Ig, sLAG-3 D1D4-Ig, or IMP321);

ii) domains D1-D4 of LAG-3, linked to Ig Fc sequence by a second linker(LAG-3 D1D4-linker2-Ig, or sLAG-3 D1D4-llinkerB-Ig);

iii) domains D1 and D2 of LAG-3, linked to Ig Fc sequence by the secondlinker (LAG-3 D1D2-linker2-Ig, or sLAG-3 D1D2-linkerB-Ig); and

iv) domains D1-D4 of LAG-3 linked to Ig Fc sequence by the first linker,but with a mutation in the MHC class II binding site of the D1 domain ofLAG-3, at position R75 (R75A), which enhances binding to MHC class IImolecules by three-fold or more (Huard et al., Proc. Natl. Acad. Sci.USA, 1997, 94:5744) (IMP321 R75A).

The derivatives are illustrated in FIG. 5.

MHC class II+ Raji cells were incubated for 45 minutes at 4° C. withvarious concentrations of the LAG-3 derivatives, or with a human IgG1antibody (hIgG1) as a negative control. The LAG-3 molecules bound to thecell surface were revealed with a FITC-conjugated goat anti-mouse Ig(Coulter). The cells were analyzed by flow cytometry. The results,expressed as fluorescence intensity units, are shown in FIG. 6. Theresults show that all of the LAG-3 derivatives bound to MHC classII-positive cells.

EXAMPLE 6

Inhibition of Binding of the LAG-3 Derivative IMP321 to MHC ClassII-Positive Cells by Antibodies that Block Binding of LAG-3 to MHC ClassII Molecules

17B4 and 11E3 are anti-LAG-3 monoclonal antibodies that are known toblock binding of LAG-3 to MHC class II molecules. Binding of anIMP321-label conjugate (LAG-3Ig-Alexa 488) to MHC class II-positive Bcells (Raji cells) was determined following pre-incubation of theconjugate (4 μg/ml at 4° C.) with 17B4 or 11E3 blocking antibody, orwith an isotype-matched negative control monoclonal antibody (mIgG1).Analysis of cell-bound fluorescence was carried out usingfluorescence-activated cell sorting (FACS). The results are shown inFIG. 7.

The results show that binding of IMP321 to Raji cells was inhibited byLAG-3-specific monoclonal antibody that blocks binding of LAG-3 to MHCclass II molecules.

EXAMPLE 7

Activation of Monocytes by LAG-3 Derivatives

THP-1 cells were incubated for 4 hours at 4° C. with the LAG-3derivatives illustrated in FIG. 5, or with human IgG1 as a negativecontrol. The amount of secretion by the THP-1 cells of the chemokineCCL4, and the cytokine Tumor Necrosis Factor-α, TNF-α, was determined,and was used as a measure of monocyte activation. CCL4 and TNF-αsecretion was quantified in the cell supernatants using a CytometricBeads Array. The results of the CCL4 determinations are shown in FIG. 8,and the results of the TNF-α determinations are shown in FIG. 9.

The results show that the LAG-3 derivates were all able to activateTHP-1 monocytic cells.

EXAMPLE 8

Inhibition of IMP321-Induced Monocyte Activation by Antibodies thatBlock Binding of LAG-3 to MHC Class II Molecules

IMP321 (20 ng/ml) was preincubated with 17B4 or 11E3 antibody (at +37°C.), before incubation of the mixture with THP-1 cells for 4 hours. Theamount of CCL4 secretion by the THP-1 cells was used to determine thelevel of monocyte activation. The results of two experiments are shownin FIG. 10.

The results demonstrate that IMP321-induced monocyte activation isinhibited by the blocking anti-LAG-3 mAbs 17B4 and 11E3. This indicatesthat the ability of IMP321 to activate monocytes is dependent on bindingof IMP321 to MHC class II molecules.

EXAMPLE 9

Activation of Primary Antigen-Presenting Cells (APCs) by LAG-3Derivatives

Human peripheral blood mononuclear cells (PBMCs) were incubated for 4hours with the LAG-3 derivatives illustrated in FIG. 5, or with humanIgG1 as a negative control, in the presence of brefeldin, a secretioninhibitor. The cytokine response of the APCs present in the PBMCs wasdetermined by intracellular staining of CCL4, a chemokine known tofavour the Th1 and CD8-positive response, and TNF-α, a multifunctionalcytokine which directly inhibits tumorigenesis. The results wereanalyzed by cytometry. The results, represented by the percentage ofcells expressing CCL4 and/or TNF-α in MHC class II-positive cells, areshown in FIG. 11.

The results show that all the LAG-3 derivatives tested induced theproduction of CCL4, and TNF-α.

EXAMPLE 10

Activation of T Cells by LAG-3 Derivatives

Human PBMCs were incubated for 18 hours with the LAG-3 derivativesillustrated in FIG. 5, or with human IgG1 as a negative control.Brefeldin was present for the last 16 hours of the incubation. Thecytokine response of CD8-positive T cells after 18 hour exposure toLAG-3 derivatives was followed by intracellular staining of CCL4, IFN-γand TNF-α and analyzed by cytometry. The results, represented as thepercentage of cells expressing CCL4, IFN-γ and/or TNF-α inCD3-positive/CD8-positive T cells, are shown in FIG. 12.

The results show that all of the LAG-3 derivatives tested inducedactivation of Type 1 cytotoxic CD8-positive T cells (Tc1 cells). It isconcluded that, through binding to MHC class II molecules expressed byAPCs, the LAG-3 derivatives induced activation of Tc1 cells. Activationof Tc1 cells forms the main anti-tumor immune response.

1.-29. (canceled)
 30. A combined preparation for enhancing a reductionof tumor growth, which comprises: (a) a derivative of LAG-3 protein thatis able to bind to MHC class II molecules, wherein the derivativecomprises: a 30 amino acid extra-loop sequenceGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 2) of domain D1 of humanLAG-3 protein; or a variant of the 30 amino acid extra-loop sequenceGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 of human LAG-3protein, wherein the variant comprises one or more conservative aminoacid substitutions, and has at least 70% amino acid identity with the 30amino acid extra-loop sequence; and (b) an anti-neoplastic agent,wherein the anti-neoplastic agent is a platinum-based anti-neoplasticagent or a topoisomerase I inhibitor, the derivative and theanti-neoplastic agent causing a synergistic reduction of tumor growthwhen administered to a subject in need thereof, the synergisticreduction of tumor growth being more than a sum of a reduction of tumorgrowth caused by administration to the subject of the derivative aloneand the anti-neoplastic agent alone.
 31. A combined preparationaccording to claim 30, wherein the derivative comprises an amino acidsequence that has at least 90% amino acid identity with domain D1 andoptionally D2 of LAG-3 protein.
 32. A combined preparation according toclaim 30, wherein the derivative comprises an amino acid sequence thathas at least 90% amino acid identity with domains D1, D2, and D3 andoptionally D4 of LAG-3 protein.
 33. A combined preparation according toclaim 30, wherein the derivative is fused to an Immunoglobulin Fcsequence.
 34. A combined preparation according to claim 30, wherein thederivative of LAG-3 protein is recombinant soluble human LAG-3Ig fusionprotein (IMP321).
 35. A combined preparation according to claim 30,wherein the anti-neoplastic agent is selected from the group consistingof oxaliplatin, carboplatin, and topotecan.
 36. A pharmaceuticalcomposition for enhancing a reduction of tumor growth, which comprises:(a) a derivative of LAG-3 protein that is able to bind to MHC class IImolecules, wherein the derivative comprises: a 30 amino acid extra-loopsequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 ofhuman LAG-3 protein; or a variant of the 30 amino acid extra-loopsequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 ofhuman LAG-3 protein, wherein the variant comprises one or moreconservative amino acid substitutions, and has at least 70% amino acididentity with the 30 amino acid extra-loop sequence; (b) ananti-neoplastic agent, wherein the anti-neoplastic agent is aplatinum-based anti-neoplastic agent or a topoisomerase I inhibitor; and(c) a pharmaceutically acceptable carrier, excipient, or diluent, thederivative and the anti-neoplastic agent causing a synergistic reductionof tumor growth when administered to a subject in need thereof, thesynergistic reduction of tumor growth being more than a sum of areduction of tumor growth caused by administration to the subject of thederivative alone and the anti-neoplastic agent alone.
 37. Apharmaceutical composition according to claim 36, wherein the derivativeof LAG-3 protein comprises an amino acid sequence that has at least 90%amino acid identity with domain D1 and optionally D2 of LAG-3 protein.38. A pharmaceutical composition according to claim 36, wherein thederivative comprises an amino acid sequence that has at least 90% aminoacid identity with domains D1, D2, D3 and optionally D4 of LAG-3protein.
 39. A pharmaceutical composition according to claim 36, whereinthe derivative is fused to an Immunoglobulin Fc sequence.
 40. Apharmaceutical composition according to claim 36, wherein the derivativeof LAG-3 protein is recombinant soluble human LAG-3Ig fusion protein(IMP321).
 41. A pharmaceutical composition according to claim 36,wherein the anti-neoplastic agent is selected from the group consistingof oxaliplatin, carboplatin, and topotecan.
 42. A method of treating orameliorating a cancer, which comprises: administering to a subject inneed of such treatment or amelioration an effective amount of: aderivative of LAG-3 protein that is able to bind to MHC class IImolecules, wherein the derivative comprises: a 30 amino acid extra-loopsequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO: 2) of domain D1 ofhuman LAG-3 protein; or a variant of the 30 amino acid extra-loopsequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 ofhuman LAG-3 protein, wherein the variant comprises one or moreconservative amino acid substitutions, and has at least 70% amino acididentity with the 30 amino acid extra-loop sequence; and ananti-neoplastic agent, wherein the anti-neoplastic agent is aplatinum-based anti-neoplastic agent or a topoisomerase I inhibitor; andsynergistically reducing tumor growth in the subject, the synergisticreduction of tumor growth being more than a sum of a reduction of tumorgrowth caused by administration to the subject of the derivative aloneand the anti-neoplastic agent alone.
 43. A method according to claim 42,wherein the derivative and the anti-neoplastic agent are administeredsequentially to the subject.
 44. A method according to claim 42, whereinthe derivative is administered after the anti-neoplastic agent.
 45. Amethod according to claim 43, wherein the derivative and theanti-neoplastic agent are administered to the subject within 96 hours ofeach other.
 46. A method according to claim 42, wherein the derivativeand the anti-neoplastic agent are co-administered to the subject.
 47. Amethod according to claim 42, wherein the derivative is administered tothe subject at a dose which is a molar equivalent of 0.25-30 mg ofLAG-3Ig fusion protein IMP321 per individual.
 48. A method according toclaim 42, wherein a plurality of doses of the derivative is administeredto the subject.
 49. A method according to claim 42, wherein a pluralityof doses of the anti-neoplastic agent is administered to the subject.50. A method according to claim 42, wherein a dose of the derivative isadministered before, with, or after each administration of two or moredoses of the anti-neoplastic agent.
 51. A method according to claim 42,wherein the derivative of LAG-3 protein comprises an amino acid sequencethat has at least 90% amino acid identity with domain D1 and optionallyD2 of human LAG-3 protein.
 52. A method according to claim 42, whereinthe derivative comprises an amino acid sequence that has at least 90%amino acid identity with domains D1, D2, D3 and optionally D4 of humanLAG-3 protein.
 53. A method according to claim 42, wherein thederivative is fused to an Immunoglobulin Fc sequence.
 54. A methodaccording to claim 42, wherein the derivative of LAG-3 protein isrecombinant soluble human LAG-3Ig fusion protein (IMP321).
 55. A methodaccording to claim 42, wherein the anti-neoplastic agent is selectedfrom the group consisting of oxaliplatin, carboplatin, and topotecan.56. A method of treating or ameliorating a cancer in a subjectadministered an anti-neoplastic agent, and in need thereof, wherein theanti-neoplastic agent is a platinum-based anti-neoplastic agent or atopoisomerase I inhibitor, which comprises: administering an effectiveamount of a derivative of LAG-3 protein that is able to bind to MHCclass II molecules to the subject, wherein the derivative comprises: a30 amino acid extra-loop sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ IDNO:2) of domain D1 of human LAG-3 protein; or a variant of the 30 aminoacid extra-loop sequence GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) ofdomain D1 of human LAG-3 protein, wherein the variant comprises one ormore conservative amino acid substitutions, and has at least 70% aminoacid identity with the 30 amino acid extra-loop sequence; andsynergistically reducing tumor growth in the subject, the synergisticreduction of tumor growth being more than a sum of a reduction of tumorgrowth caused by administration to the subject of the derivative aloneand the anti-neoplastic agent alone.
 57. A method according to claim 56,wherein the derivative of LAG-3 protein comprises an amino acid sequencethat has at least 90% amino acid identity with domain D1 and optionallyD2 of LAG-3 protein.
 58. A method according to claim 56, wherein thederivative comprises an amino acid sequence that has at least 90% aminoacid identity with domains D1, D2, D3 and optionally D4 of LAG-3protein.
 59. A method according to claim 56, wherein the derivative ofLAG-3 protein is recombinant soluble human LAG-3Ig fusion protein(MP321).
 60. A method according to claim 56, wherein the anti-neoplasticagent is selected from the group consisting of oxaliplatin, carboplatin,and topotecan.
 61. A method of treating or ameliorating a cancer in asubject administered a derivative of LAG-3 protein that is able to bindto MHC class II molecules, and in need thereof, wherein the derivativecomprises: a 30 amino acid extra-loop sequenceGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 of human LAG-3protein; or a variant of the 30 amino acid extra-loop sequenceGPPAAAPGHPLAPGPHPAAPSSWGPRPRRY (SEQ ID NO:2) of domain D1 of human LAG-3protein, wherein the variant comprises one or more conservative aminoacid substitutions, and has at least 70% amino acid identity with the 30amino acid extra-loop sequence, which comprises: administering aneffective amount of an anti-neoplastic agent to the subject, wherein theanti-neoplastic agent is a platinum-based anti-neoplastic agent or atopoisomerase I inhibitor; and synergistically reducing tumor growth inthe subject, the synergistic reduction of tumor growth being more than asum of a reduction of tumor growth caused by administration to thesubject of the derivative alone and the anti-neoplastic agent alone. 62.A method according to claim 61, wherein the derivative of LAG-3 proteincomprises an amino acid sequence that has at least 90% amino acididentity with domain D1 and optionally D2 of LAG-3 protein.
 63. A methodaccording to claim 61, wherein the derivative comprises an amino acidsequence that has at least 900% amino acid identity with domains D1, D2,D3 and optionally D4 of LAG-3 protein.
 64. A method according to claim61, wherein the derivative of LAG-3 protein is recombinant soluble humanLAG-3Ig fusion protein (IMP321).
 65. A method according to claim 61,wherein the anti-neoplastic agent is selected from the group consistingof oxaliplatin, carboplatin, and topotecan.